Bifidobacterium animalis and application of compound bacterium preparation prepared from bifidobacterium animalis in preparing medicine for treating or preventing avian influenza virus infection

ABSTRACT

Bifidobacterium animalis and application of a compound bacterium preparation prepared from the Bifidobacterium animalis in the preparation of a medicine for treating or preventing avian influenza virus infection. The invention discovers that Bifidobacterium animalis ATCC Accession No. 25527 can be used for treating animals infected by H7N9, and after the Bifidobacterium animalis ATCC Accession No. 25527 is compounded with Bifidobacterium pseudolongum ATCC Accession No. 25526, the effect is better. The compound preparation can regulate the body immune response, remarkably improve the weight loss and lung tissue injury caused by H7N9 influenza virus infection, and obviously improve the survival rate of mice infected by H7N9 influenza virus. The compound probiotic preparation provided by the invention has a remarkable effect of resisting H7N9 influenza virus infection, and can be effectively used for preventing and treating avian influenza virus infection.

CROSS-REFERENCE TO RELATED APPLICATIONS

The subject application claims priority on Chinese Application No.CN202010031196.6 filed on Jan. 13, 2020 in China. The contents andsubject matter of the Chinese priority application is incorporatedherein by reference.

BACKGROUND OF THE INVENTION Technical Field

The invention relates to the field of biological preparations againstinfluenza virus infections, in particular to Bifidobacterium animalisand an application of a compound bacterium preparation prepared from theBifidobacterium animalis in preparation of a medicine for treating orpreventing avian influenza virus infection.

Description of Related Art

Avian influenza virus belongs to the Orthomyxoviridae Influenza A virusgenus and is a highly contagious infectious disease worldwide. Thedisease mainly occurs in poultry and wild birds, seriously hindering thedevelopment of the poultry industry. Moreover, some of its subtypes (H5,H7, H9, H10) can also transmit to people cross-species, causing fever,cough, muscle aches, conjunctivitis, respiratory diseases and othersymptoms, and even death in severe cases, posing a serious threat tohuman health.

The avian influenza virus has always been relatively mild and onlyspreads among animals. However, human cases of H7N9 influenza were firstdetected in Shanghai and Anhui in March 2013, proving that this virus isable to spread from poultry to humans across species isolation. Poultryhosts infected with H7N9 include chickens, ducks, geese, and quails, andwild hosts include waterfowls around rivers, lakes, and seas, as well asvarious migratory wild birds. Close contact with infected poultry, itssecretions and excrement, or the virus-contaminated environment cancause human infection with H7N9 avian influenza virus. According tostatistics from the World Health Organization (WHO), as of March 2018,the number of people infected with H7N9 worldwide has reached 1,564.Among them, a total of 1,438 cases and 570 deaths have been reported inChina, with a fatality rate high up to 39.63%. Therefore, wellprevention and control of avian influenza H7N9 can not only promote thehealthy development of the livestock and poultry breeding industry, butalso effectively maintain social and economic stability and ensurepublic health safety.

At present, the prevention and control technologies for avian influenzaat home and abroad mainly include: culling, disinfection, biologicalsafety, and vaccination. Although culling infected livestock and poultrycan curb the spread of avian influenza to a certain extent, wild birds,as another important host of avian influenza, are difficult to controlthrough culling. Moreover, large-scale culling of livestock and poultrywill also cause huge economic losses. Another effective measure toprevent the occurrence of avian influenza is vaccination. When thevaccine strain is the same as the clinically epidemic strain, the idealprotective effect can be obtained, but when the vaccine strain isdifferent from the epidemic strain, it cannot provide effective immuneprotection. There are many serotypes of the avian influenza virus, andthe mixed infection of influenza A viruses of different subtypes oftenoccurs genetic recombination to form new avian influenza viruses, whichbrings great difficulties to the development and application ofvaccines. Immunization failures caused when vaccines used are differentfrom epidemic strains often occur in production practice.

Therefore, in the process of preventing avian influenza, in addition tostrengthening vaccine development, other biological preparations withhighly effective anti-flu effects should also be actively developed.Bifidobacterium are generally considered to be probiotics. When asufficient amount of Bifidobacteria is ingested, Bifidobacteria canpromote the health of the body. It was reported in references that theintake of Bifidobacterium could improve the gastrointestinalenvironment, prevent and treat some diseases caused by intestinalmicroecological disorders, including gastrointestinal infections,constipation, irritable bowel syndrome (IBS), inflammatory bowel disease(IBD)), ulcerative colitis, allergies, antibiotic-induced diarrhea,cardiovascular disease, and colorectal cancer. It was reported by AlvinIbarra et al. (2018) that in a randomized double-blind andplacebo-controlled trial, through the evaluation of various relatedindicators, supplementation of Bifidobacterium animalis could improvestool frequency. It was found by Guo Huihui et al. (2017) thatBifidobacterium could effectively inhibit the decrease in bloodhemoglobin concentration in mice caused by ulcerative colitis and theshortening of colon length caused by ulcerative colitis, and thatBifidobacterium took a therapeutic effect on ulcerative colitis byadjusting the intestinal microecological balance. Gloria Solano-Aguilaret al. (2018) reported that feeding subsp. Lactis of Bifidobacteriumanimalis had a local immunomodulatory effect on piglets infected withAscaris suum. During nematode infection, by reducing the components oftype 2 allergic reactions in pigs, it changed the local immune responseand improved intestinal function. Jung Min Chae et al., (2018) reportedthat subsp. lactis BB12 of Bifidobacterium animalis could significantlyimprove DSS-induced colitis, and suggested that probiotic BB12 couldimprove the survival rate of intestinal cells by regulating theexpression of pro-apoptotic cytokines. Hüseyin Sancar Bozkurt et al.,(2019) reported that Bifidobacteria could be used for the prevention ofcolorectal cancer.

Although there are some reports on the prevention and treatment ofintestinal infections by Bifidobacterium, there are few reports onBifidobacterium as probiotic preparations against intestinal infections,especially against respiratory tract infections. At present, there is noreport about the antiviral effect of Bifidobacterium animalis. Theinvention reports the application of Bifidobacterium animalis againstinfluenza virus for the first time.

BRIEF SUMMARY OF THE INVENTION

The invention is directed to the difficulty about prevention andtreatment of influenza virus infection, and intended to provide theapplication of Bifidobacterium animalis having American Type CultureCollection (ATCC) Accession No. 25527 in the preparation of a medicinefor treating or preventing avian influenza virus infection.

Another objective of the invention is to provide a medicine for treatingor preventing avian influenza virus infection. The medicine includesBifidobacterium animalis having ATCC Accession No. 25527 andBifidobacterium pseudolongum having ATCC Accession No. 25526.

The final objective of the invention is to provide the application of apreparation comprising Bifidobacterium animalis having ATCC AccessionNo. 25527 and Bifidobacterium pseudolongum having ATCC Accession No.25526 in the preparation of a medicine for treating or preventing avianinfluenza virus infection.

In order to achieve the above objectives, the invention adopts thefollowing technical measures:

The application of Bifidobacterium animalis ATCC Accession No. 25527 inthe preparation of a medicine for treating or preventing avian influenzavirus infection, including the use of ATCC Accession No. 25527 as thesole active ingredient or one of active ingredients for the preparationof a medicine for treating or preventing diseases with avian influenzavirus infection.

In the above-mentioned application, preferably, the effective bacterialconcentration of the Bifidobacterium animalis is ≥1×10⁹ CFU/ml.

A medicine for treating or preventing avian influenza virus infection,including Bifidobacterium animalis ATCC Accession No. 25527 andBifidobacterium pseudolongum ATCC Accession No. 25526;

In the above-mentioned medicine, preferably, the ratio of the effectivebacterial concentration of the Bifidobacterium animalis ATCC AccessionNo. 25527 to the Bifidobacterium pseudolongum ATCC Accession No. 25526is 1:0.5-2.

In the above-mentioned medicine, the optimal ratio of effectivebacterial concentration of Bifidobacterium animalis ATCC Accession No.25527 to Bifidobacterium pseudolongum ATCC Accession No. 25526 is 1:1,and the effective bacterial concentration after the mixture ofBifidobacterium animalis ATCC Accession No. 25527 and Bifidobacteriumpseudolongum ATCC Accession No. 25526 is ≥1×10⁹ CFU/ml.

The application of the preparation including ATCC Accession No. 25527and ATCC Accession No. 25526 in the preparation of a medicine fortreating and preventing avian influenza virus infection also belongs tothe protection scope of the invention;

The preparation including ATCC Accession No. 25527 and ATCC AccessionNo. 25526 can also be used to prepare interferon stimulants after avianinfluenza virus infection.

The symptoms of the infection described above include weight loss,death, pathological injury to the lungs, and the proliferation of theavian influenza virus in the lungs due to the avian influenza virusinfection.

Oral administration of Bifidobacterium animalis ATCC Accession No. 25527to treat or prevent avian influenza virus infection and oraladministration of Bifidobacterium animalis ATCC Accession No. 25527 andBifidobacterium pseudolongum ATCC Accession No. 25526 to treat orprevent avian influenza virus infection also fall within the protectionscope of the invention.

Oral administration of Bifidobacterium animalis ATCC Accession No. 25527to treat weight loss, death, and lung pathological injury caused byavian influenza virus infection, or the proliferation of H7N9 influenzavirus in the lungs;

Oral administration of Bifidobacterium animalis ATCC Accession No. 25527and Bifidobacterium pseudolongum ATCC Accession No. 25526 to treatweight loss, death, and lung pathological injury caused by H7N9infection, or proliferation of avian influenza virus in the lungs.

The avian influenza virus described above is preferably H7N9.

Compared with the prior art, the invention has the following advantages:

(1) The compound probiotic preparation of the invention can regulate theimmune response of the host body, promote the expression of multiplecytokines in the early stage of avian influenza virus infection, andincrease the body's elimination of the virus; reduce the expression ofcertain cytokines in the middle of infection and reduce the inflammatoryinjury caused by overexpression of cytokines.

(2) The probiotic preparation of the invention can quickly improve theability of the body to produce interferon and clear the virus in timeafter the animal body is infected with avian influenza virus.

(3) The compound probiotic preparation of the invention cansignificantly inhibit the proliferation of H7N9 influenza virus in mice,improve the weight loss caused by H7N9 infection, and improve thesurvival rate of infected mice. Therefore, the compound probioticpreparation has obvious resistance to H7N9 influenza virus infection.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIGS. 1A and 1B show the colony morphology of the Bifidobacterium usedin the present invention, where FIG. 1A shows the colony morphology ofBifidobacterium pseudolongum; and FIG. 1B shows the colony morphology ofBifidobacterium animalis.

FIG. 2 shows the effect of intragastric administration of the probioticpreparation of the present invention on the survival rate of SPF miceinfected with H7N9, where the vertical axis shows the percentage ofsurvival.

FIG. 3 shows the effect of intragastric administration of the probioticpreparation of the present invention on the weight of SPF mice infectedwith H7N9, where the vertical axis shows the percentage of the initialweight.

FIG. 4 shows the effect of intragastric administration of the probioticpreparation of the present invention on the survival rate of germ-freemice infected with H7N9, where the vertical axis shows the percentage ofsurvival.

FIG. 5 shows the effect of intragastric administration of the probioticpreparation of the present invention on the proliferation of lung virusin SPF mice infected with H7N9, where the vertical axis shows the virustiter (Log₁₀ EID₅₀/ml).

FIGS. 6A to 6L show the effect of intragastric administration of theprobiotic preparation of the present invention on pathological changesin the lungs of SPF mice infected with H7N9, where FIG. 6A show theeffect in Day 0 with PBS; FIG. 6B shows the effect in Day 0 withBifidobacterium pseudolongum; FIG. 6C shows the effect in Day 0 withBifidobacterium animalis; FIG. 6D shows the effect in Day 0 withBifidobacterium pseudolongum and Bifidobacterium animalis; FIG. 6E showthe effect in Day 3 with PBS; FIG. 6F shows the effect in Day 3 withBifidobacterium pseudolongum; FIG. 6G shows the effect in Day 3 withBifidobacterium animalis; FIG. 6H shows the effect in Day 3 withBifidobacterium pseudolongum and Bifidobacterium animalis; FIG. 6I showthe effect in Day 5 with PBS; FIG. 6J shows the effect in Day 5 withBifidobacterium pseudolongum; FIG. 6K shows the effect in Day 5 withBifidobacterium animalis; and FIG. 6L shows the effect in Day 5 withBifidobacterium pseudolongum and Bifidobacterium animalis.

FIGS. 7A to 7F show the effect of intragastric administration of theprobiotic preparation of the present invention on the changes of lungcytokines in SPF mice infected with H7N9, where FIG. 7A shows the effecton TNF-α (pg/mL) as represented by the vertical axis; FIG. 7B shows theeffect on IL-10 (pg/mL) as represented by the vertical axis; FIG. 7Cshows the effect on IL-1β (pg/mL) as represented by the vertical axis;FIG. 7D shows the effect on IFN-γ (pg/mL) as represented by the verticalaxis; FIG. 7E shows the effect on IL-6 (pg/mL) as represented by thevertical axis; and FIG. 7F shows the effect on IFN-β (pg/mL) asrepresented by the vertical axis.

FIGS. 8A to 8F show the effect of intragastric administration of theprobiotic preparation of the present invention on the changes of serumcytokines in SPF mice infected with H7N9, where FIG. 8A shows the effecton TNF-α (pg/mL) as represented by the vertical axis; FIG. 8B shows theeffect on IL-10 (pg/mL) as represented by the vertical axis; FIG. 8Cshows the effect on IL-1β (pg/mL) as represented by the vertical axis;FIG. 8D shows the effect on IFN-γ (pg/mL) as represented by the verticalaxis; FIG. 8E shows the effect on IL-6 (pg/mL) as represented by thevertical axis; and FIG. 8F shows the effect on IFN-β (pg/mL) asrepresented by the vertical axis.

DETAILED DESCRIPTION OF THE INVENTION

The experimental methods and conditions in the following examples areconventional methods unless otherwise specified. These examples are onlyused to illustrate the invention, and the protection scope of theinvention is not limited by these examples. The technical solutions ofthe invention, unless otherwise specified, are conventional solutions inthe field; unless otherwise specified, the reagents or materials are allsourced from commercial channels.

The invention takes avian influenza virus H7N9 as an example toillustrate the application of Bifidobacterium animalis ATCC AccessionNo. 25527 and its compound preparation in the preparation of a medicinefor preventing or treating avian influenza virus infection.

The following specific embodiments further illustrate the invention:

Example 1

Preparation Method of a Probiotic Preparation Against H7N9 AvianInfluenza Virus Infection:

1. Preparation of Single-Bacterium Preparations of Bifidobacteriumanimalis and Bifidobacterium pseudolongum

Bifidobacterium animalis ATCC Accession No. 25527 and Bifidobacteriumpseudolongum ATCC Accession No. 25526. The lyophilized powder of thestrain was resuspended with germ-free PBS, an appropriate count ofbacteria was picked up with an inoculating loop to streak and rejuvenateon an MRS solid plate medium, and cultivated under an anaerobiccondition at 37° C. for 24-48 hours. The rejuvenated Bifidobacteriumanimalis and Bifidobacterium pseudolongum were respectively cultured inMRS streaks (FIG. 1A, FIG. 1B). After culturing for 24 hours under ananaerobic condition at 37° C., bacterial lawns on the MRS medium platewere washed with PBS to collect the bacterial suspension. The bacterialsuspension was centrifuged at 4° C., 5000 rpm for 10 minutes, thesupernatant was discarded, the collected Bifidobacterium animalis andBifidobacterium pseudolongum precipitate were resuspended in PBS, andviable bacteria were counted on the MRS plate. The concentration of thebacterial suspension was adjusted so that the number of viable bacteriais 1×10⁹ CFU/ml, and the resulting products were stored assingle-bacterium preparations against influenza virus at 4° C. for lateruse.

2. Preparation Method of Compound Probiotic Preparation Against H7N9Avian Influenza Virus Infection

The single-bacterium preparations prepared by the above method weremixed at a ratio of 1:1 (V/V) to prepare a compound probioticpreparation. The concentrations of Bifidobacterium animalis ATCCAccession No. 25527 and Bifidobacterium pseudolongum ATCC Accession No.25526 in the compound probiotic preparation were both 5×10⁸ CFU/ml, andthe total bacterial cell concentration was 1×10⁹ CFU/ml.

The single and compound bacterium probiotic preparations in Example 1were used in the following examples.

Example 2

The effect of the probiotic preparation prepared in Example 1 on themortality and weight of mice after H7N9 avian influenza virus infection

1. Effect on the Mortality and Weight of SPF Mice

The test animals were 8-week-old female C57BL/6 SPF(Specific-Pathogen-Free) mice, in a total of 80, randomly divided into 4groups, 20 in each group, and all the animals were raised in an ABSL3(Animal Biological Safety Level-3) laboratory. The first group was agerm-free PBS control group in which each mice took 100 ul byintragastric administration; the second group was a Bifidobacteriumpseudolongum ATCC Accession No. 25526 single-bacterium preparation groupin which each mice took 100 ul by intragastric administration; the thirdgroup is a Bifidobacterium animalis ATCC Accession No. 25527single-bacterium preparation group in which each mice took 100 ul byintragastric administration; the fourth group is a Bifidobacteriumanimalis ATCC Accession No. 25527-Bifidobacterium pseudolongum ATCCAccession No. 25526 compound preparation group in which each mice took100 ul by intragastric administration.

Antibiotic treatment: Before intragastric administration of theprobiotic preparations to test mice, the mice were treated with acompound antibiotic solution. The compound antibiotic preparation,including ampicillin (1 mg/ml), streptomycin (5 mg/ml), vancomycin (0.25mg/ml), colistin (1 mg/ml), was added for 3 consecutive days to thegerm-free water the mice drank, and the addition of the compoundantibiotic preparation was stopped one day before the intragastricadministration of a probiotic preparation. All antibiotics werepurchased with sigma-Aldrich company.

After intragastric administration of PBS or the probiotic preparationsto the mice, all mice were infected with one LD50 H7N9 avian influenzavirus intranasally. After influenza virus infection, the mice wereobserved and weighed for 15 consecutive days, and the survival statuswas recorded.

The results showed that compared with the survival rate of the controlgroup taking intragastric administration of PBS, the survival rate ofSPF mice taking intragastric administration of Bifidobacterium animalissingle-bacterium preparation (P=0.0259) and the survival rate of SPFmice taking the Bifidobacterium animalis-Bifidobacterium pseudolongumcompound preparation (P=0.0015)) can both be significantly increased by20% and 40% respectively after the mice were infected with H7N9 avianinfluenza virus; however, the intragastric administration of theBifidobacterium pseudolongum single-bacterium preparation has no obviouseffect on mice infected with H7N9 influenza virus (Table 1, FIG. 2).

TABLE 1 Effect of the probiotic preparations on the survival rate of SPFmice infected with H7N9 influenza virus B. animalis + Group PBS controlB. pseudolongum B. animalis B. pseudolongum Number at time of 20 20 2020 infection Number of survivals 4 4 8 12 15 days post infectionSurvival rate (%) 20 20 40 60 Mortality 80 80 60 40

In terms of weight, mice taking intragastric administration of theBifidobacterium animalis-Bifidobacterium pseudolongum compoundpreparation were significantly improved in weight loss 6-10 days afterbeing infected with H7N9 influenza virus. Mice taking the intragastricadministration of the Bifidobacterium pseudolongum single-bacteriumpreparation were significantly improved in weight loss to a certainextent 6-10 days after being infected with H7N9 influenza virus (Table2, FIG. 3).

TABLE 2 Effect of the probiotic preparations on the weight of SPF miceinfected with H7N9 influenza virus Weight percentage (%) BifidobacteriumB. animalis + Number PBS control B. pseudolongum animalis B.pseudolongum of days group group group group 0 100 100 100 100 1 99.95 ±1.27 98.21 ± 5.40 99.98 ± 2.04 98.36 ± 3.61 2 99.66 ± 1.34 99.23 ± 4.4099.69 ± 2.76 98.18 ± 4.98 3 100.64 ± 1.81  101.79 ± 3.00  99.05 ± 3.8896.91 ± 4.43 4 100.30 ± 2.90  99.99 ± 2.01 97.08 ± 4.13 96.20 ± 5.33 597.43 ± 2.88 99.26 ± 2.54 96.09 ± 4.03 96.69 ± 5.94 6 92.58 ± 3.37 94.57± 4.86 93.42 ± 4.59 97.14 ± 6.82 7 85.31 ± 2.37 87.53 ± 3.97 90.96 ±5.46 95.95 ± 6.31 8 82.53 ± 2.88 84.99 ± 4.49 89.18 ± 5.88 95.18 ± 6.389 85.71 ± 6.87 85.71 ± 8.75 88.03 ± 5.64 93.45 ± 7.90 10 94.78 ± 1.97 92.49 ± 10.29 89.71 ± 5.11  92.22 ± 11.51 11 100.81 ± 4.04  97.43 ±9.25 93.68 ± 3.92  95.59 ± 11.95 12 101.38 ± 5.26  97.86 ± 6.02 97.38 ±4.48 98.89 ± 9.07 13 101.93 ± 4.72  98.98 ± 3.94 98.25 ± 3.95 100.02 ±8.44  14 101.41 ± 2.08  101.83 ± 4.38  100.18 ± 2.73  101.36 ± 8.73  15101.09 ± 2.62  102.61 ± 4.34  101.37 ± 2.78  102.06 ± 8.60  Note: Thenumber of days is the number of days after the mouse is infected withinfluenza virus.

2. Effect on Mortality of Germ-Free Mice

According to the effects of the aforementioned probiotic preparations onthe mortality and weight of SPF mice infected with H7N9 influenza virus,19 germ-free mice were used in this test to detect the effects of theprobiotic preparations in Example 1 on germ-free mice infected with H7N9avian influenza virus; the 19 germ-free mice were randomly divided into3 groups. The first group of 6 mice was a germ-free PBS control group inwhich each mice took 100 ul by intragastric administration; the secondgroup of 6 mice was a Bifidobacterium animalis ATCC Accession No. 25527single-bacterium preparation group in which each mice took 100 ul byintragastric administration; and the third group of 7 mice was aBifidobacterium animalis ATCC Accession No. 25527-Bifidobacteriumpseudolongum ATCC Accession No. 25526 compound preparation group inwhich each mice took 100 ul by intragastric administration. Antibiotictreatment was the same as that for SPF mice. One day after intragastricadministration of PBS or the probiotic preparations to the mice, all themice were intraperitoneally injected with 0.5 LD50 dose of H7N9 avianinfluenza virus. After infection, the status of germ-free mice wasobserved for 15 consecutive days and the survival status was recorded.

According to the effect of the probiotic preparations of the inventionon the survival rate of SPF mice infected with H7N9 influenza virus, theintragastric administration of the Bifidobacterium animalissingle-bacterium preparation and the compound preparation cansignificantly improve the survival rate of the mice. In order to furthereliminate the influence of microorganisms remaining in the intestinaltract after antibiotic treatment, germ-free mice were used for animalexperiments in this example. The results also showed that the survivalrates of germ-free mice taking the intragastric administration of theBifidobacterium animalis single-bacterium preparation (P=0.0201) andtaking intragastric administration of Bifidobacteriumanimalis-Bifidobacterium pseudolongum compound preparation (P=0.0323)were both improve significantly by 50% and 40.47% respectively ascompared with that of the PBS control group. Through comparison of theresults of SPF mice and germ-free mice, the only difference is that inthe germ-free mice, the protective effect of the intragastricadministration of Bifidobacterium animalis (66.67%) was even slightlyhigher than that of the compound preparation (57.14%), but in the SPFmice, the protective effect for the group taking the intragastricadministration of Bifidobacterium animalis was only half of that for thegroup taking the intragastric administration of the compoundpreparation. These results showed that the intragastric administrationof Bifidobacterium animalis to animals could enhance their resistance toH7N9 influenza virus infection. For non-germ-free animals, additionaluse of Bifidobacterium pseudolongum could enhance this protective effect(Table 3, FIG. 4).

TABLE 3 Effect of the probiotic preparations on the survival rate ofgerm-free mice infected with H7N9 influenza virus Bifidobacterium B.animalis + PBS control animalis B. pseudolongum Group group group groupNumber at the time of 6 6 7 infection Number of survivals 15 1 4 4 dayspost infection Survival rate (%) 16.67 66.67 57.14 Mortality 83.33 33.3342.86

Example 3

The effect of the probiotic preparations prepared in Example 1 on thecontent of influenza virus in the lungs, the structure of lung tissue,and the content of lung cytokines and blood cytokines in mice infectedwith H7N9 avian influenza virus

96 SPF mice were randomly divided into 4 groups, 24 mice in each group.The first group was a germ-free PBS control group in which each micetook 100 ul by intragastric administration; the second group was aBifidobacterium pseudolongum ATCC Accession No. 25526 single-bacteriumpreparation group in which each mice took 100 ul by intragastricadministration; the third group is a Bifidobacterium animalis ATCCAccession No. 25527 single-bacterium preparation group in which eachmice took 100 ul by intragastric administration; the fourth group is aBifidobacterium animalis ATCC Accession No. 25527-Bifidobacteriumpseudolongum ATCC Accession No. 25526 compound preparation group inwhich each mice took 100 ul by intragastric administration.

As in Example 2, all SPF mice were raised in an ABSL3 laboratory, andsubjected to the same antibiotic treatment, intragastric administrationof probiotic preparations and H7N9 influenza virus infection.

After influenza virus infection, blood was collected on day 0, day 3,and day 5, and lung samples were taken by anatomy. There were 8 mice ineach group. Five of the eight lung samples of each group were used todetermine cytokine content and influenza virus content, and theremaining three lung samples were fixed with 4% formaldehyde solutionfor tissue section observation.

Blood sample treatment: The collected non-anticoagulated blood sampleswere rested at 4° C. overnight; after the serum was separated, the serumwas centrifuged at 4° C., 1000 rpm for 5 minutes, and the supernatantwas taken and frozen at −80° C. for detection of cytokine content.

Lung tissue treatment: the collected lung tissue samples were added with1 ml germ-free PBS (1 ml PBS/lung) and homogenized and broken by ahomogenizer, and centrifuged to take the supernatant; the supernatantwas frozen at −80° C. for detection of cytokine content and influenzavirus content.

The cytokines tested included TNF-α, IL-1β, IL-6, IL-10, IFN-γ, andIFN-β. Among them, TNF-α, IL-1β, IL-6, IL-10, and IFN-γ were detected byMagnetic Luminex® Assay multiplex kit (R & D Systems), and IFN-β wasdetected by VeriKine Mouse IFN-β enzyme-linked immunosorbent assay kit(BioLegend).

1. Table 2 Effect of the probiotic preparations on the content ofinfluenza virus in the lungs of mice infected with H7N9 avian influenzavirus: The preserved lung tissue homogenate supernatant was inoculatedinto 9-11 days old SPF chicken embryos for the determination of theinfluenza virus content in lung tissue. After the inoculated chickenembryos were incubated at 37° C. for 72 hours, and the allantoic fluidwas collected for hemagglutination test. The virus titer was calculatedusing the method described by Reed and Muench.

The test results showed that on day 3 and day 5 post H7N9 influenzavirus infection, compared with the PBS control group, the group takingintragastric administration of Bifidobacterium animalis single-bacteriumpreparation and the group taking intragastric administration ofBifidobacterium animalis-Bifidobacterium pseudolongum compoundpreparation were significantly reduced in virus content in the lungtissue; however, the intragastric administration of the Bifidobacteriumpseudolongum single-bacterium preparation had no effect on the viruscontent in the lung tissue (Table 4, FIG. 5).

TABLE 4 Effect of probiotic preparations on influenza virus content inlungs of mice infected with H7N9 avian influenza virus (log₁₀EID₅₀/ml)Bifidobacterium Bifidobacterium B. animalis + Group PBS controlpseudolongum animalis B. pseudolongum 3 days post 4.773 ± 0.446 4.650 ±0.418 3.384 ± 0.135 3.064 ± 0.447 infection 5 days post 5.380 ± 0.4605.340 ± 0.434 4.155 ± 0.450 3.768 ± 0.377 infection

2. Effect of the probiotic preparations on the structure of lung tissuein mice infected with H7N9 avian influenza virus: The results ofmicroscopic examination of lung tissue sections showed that on day 3 andday 5 post H7N9 influenza virus infection, compared with the controlgroup taking intragastric administration of PBS, the group takingintragastric administration of Bifidobacterium animalis single-bacteriumpreparation and the group taking intragastric administration ofBifidobacterium animalis-Bifidobacterium pseudolongum compoundpreparation were significantly reduced in pathological injury ofinfluenza virus infection to lung tissue; however, the intragastricadministration of the Bifidobacterium pseudolongum single-bacteriumpreparation did not improve pathological injury of influenza virusinfection to lung tissue. This further proved the anti-influenza effectof Bifidobacterium animalis (FIG. 6A to 6L).

3. Effect of the probiotic preparations on the content of lung cytokinesof mice infected with H7N9 avian influenza virus: The determinationresults of cytokine content in the supernatant of lung tissue sampletreatment showed that on day 0 post infection, there was no differencein the content of cytokines TNF-α, IL-1β, IL-6, IL-10, IFN-γ and IFN-βbetween the control group taking intragastric administration of PBS andthe groups taking intragastric administration of three probioticpreparations. On day 3 and day 5 post influenza virus infection, therewas significant difference in the content of most of the cytokinesmeasured among the group taking intragastric administration of theBifidobacterium animalis single-bacterium preparation and the grouptaking intragastric administration of the Bifidobacteriumanimalis-Bifidobacterium pseudolongum compound preparation; but on day 3and day 5 post infection, the change trend of cytokines was different.First compared with the control group, on day 3 post influenza virusinfection, the group taking intragastric administration of theBifidobacterium animalis single-bacterium preparation and the grouptaking intragastric administration of the Bifidobacteriumanimalis-Bifidobacterium pseudolongum compound preparation showed asignificant increasing trend in most of cytokines, but on day 3 and day5 post infection, the two groups showed a declining trend in cytokinecontent, and particularly the group taking intragastric administrationof the compound preparation showed a more obvious declining trend.Specifically, on day 3 post influenza virus infection, compared with thePBS control group, the group taking intragastric administration of theBifidobacterium animalis single-bacterium preparation showed an obviousincrease in the contents of IL-6, IFN-γ, and IFN-β with statisticaldifferences and showed a significant increase in the content of TNF-α;TNF-α, IL-6, IFN-γ, IFN-β increased significantly, and the group takingintragastric administration of the Bifidobacteriumanimalis-Bifidobacterium pseudolongum compound preparation showed asignificant increase in the contents of TNF-α, IL-6, IFN-γ, and IFN-βand also showed an obvious increase in the content of IL-10. On day 5post influenza virus infection, compared with the PBS control group, thegroup taking intragastric administration of the Bifidobacterium animalissingle-bacterium preparation showed an obvious decrease in the contentof IL-1β and a significant decrease in the content of IL-6, and thegroup taking intragastric administration of the Bifidobacteriumanimalis-Bifidobacterium pseudolongum compound preparation showed asignificant decrease in the contents of TNF-α, IL-10, IL-1β, and IL-6.At different measurement time points, there was no difference in thecytokine content between the group taking the Bifidobacteriumpseudolongum single-bacterium preparation and the PBS control group(Table 5, FIGS. 7A to 7F).

TABLE 5 Effect of the probiotic preparations on the content of lungcytokines of mice infected with H7N9 avian influenza virus (pg/ml)Bifidobacterium Bifidobacterium animalis B. animalis + Group PBS controlpseudolongum group B. pseudolongum Post TNF-α  2.870 ± 0.385  3.152 ±0.298  3.364 ± 0.676  3.046 ± 0.607 infection IL-1β  701.496 ± 128.655 691.518 ± 125.705  726.348 ± 131.087  706.040 ± 152.334 0 day IL-632.730 ± 5.088 34.654 ± 6.070 32.536 ± 5.301 32.454 ± 4.842 IL-10 25.124± 3.452 26.952 ± 3.663 26.490 ± 4.176 25.892 ± 4.321 IFN-γ 64.082 ±7.927 64.646 ± 8.655  66.482 ± 10.390 65.974 ± 9.104 IFN-β 76.766 ±7.213 79.460 ± 7.346 77.754 ± 7.138 78.197 ± 5.014 3 days TNF-α  8.506 ±1.796  9.246 ± 2.183 14.590 ± 2.271 14.372 ± 1.546 post IL-1β 1043.800 ±98.554   918.554 ± 139.072  985.438 ± 105.183  918.806 ± 138.435infection IL-6 138.922 ± 25.185 150.710 ± 23.596 209.614 ± 21.871229.308 ± 26.887 IL-10 28.824 ± 2.724 29.884 ± 4.120 29.898 ± 3.56634.516 ± 2.874 IFN-γ  75.224 ± 11.451  89.538 ± 15.858 101.830 ± 12.840121.928 ± 13.258 IFN-β 104.995 ± 11.718 114.274 ± 16.065 141.126 ±10.635 148.042 ± 13.554 5 days TNF-α 21.432 ± 3.714 22.844 ± 3.62719.502 ± 2.854 16.198 ± 2.173 post IL-1β 1176.910 ± 147.540 1128.034 ±173.058  923.836 ± 160.121  784.792 ± 147.886 infection IL-6 442.314 ±76.314 455.560 ± 64.557 312.492 ± 85.979 103.950 ± 25.962 IL-10 32.894 ±2.786 35.974 ± 3.910 29.846 ± 3.456 25.990 ± 2.785 IFN-γ  86.652 ±17.022  93.012 ± 17.797  89.768 ± 20.736  97.362 ± 20.056 IFN-β 197.075± 22.816 203.034 ± 27.750 209.430 ± 32.698 197.696 ± 26.924

4. Effect of the probiotic preparations on the content of serumcytokines in mice infected with H7N9 avian influenza virus: Thedetermination results of the cytokine content in mouse serum showed thaton day 0, day 3, and day 5 post H7N9 influenza virus infection, comparedwith the PBS control group, the group taking intragastric administrationof the Bifidobacterium pseudolongum preparation showed no difference inthe content of each cytokine, and only showed a significant decrease inthe content of IL-1β on day 3 post infection and a certain increase inthe content of IFN-β on day 5 post infection; however, compared with thecontrol group, the group taking intragastric administration of theBifidobacterium animalis preparation and the group intragastricadministration of the Bifidobacterium animalis-Bifidobacteriumpseudolongum compound preparation showed a significant difference in thecontents of most of cytokines on day 3 and day 5 post influenza virusinfection. On day 3 post infection, compared with the control group, thegroup taking intragastric administration of the Bifidobacterium animalispreparation showed a significant increase in the contents of TNF-α andIFN-γ and an obvious increase in the content of IFN-β; the group takingintragastric administration of the Bifidobacteriumanimalis—Bifidobacterium pseudolongum compound preparation showed asignificant increase in the contents of TNF-α, IL-10, IFN-γ, IL-6, andIFN-β but showed a significant decrease in the content of IL-1β. On day5 post infection, compared with the PBS control group, the group takingintragastric administration of the Bifidobacterium animalis preparationshowed a significant decrease in the content of IFN-γ, an obviousincrease the contents of TNF-α and IFN-β, and a significant decrease inthe content of IL-1β and IL-6, and the group taking intragastricadministration of the Bifidobacterium animalis-Bifidobacteriumpseudolongum compound preparation showed a significant increase in thecontents of IFN-γ and IFN-β and a significant decrease in the contentsof IL-1β and IL-6 (Table 6, FIGS. 8A to 8F). It can be seen that theBifidobacterium animalis-Bifidobacterium pseudolongum compoundpreparation group can significantly improve the body's ability toproduce interferon, thereby achieving the purpose of eliminating thevirus.

TABLE 6 Effect of the probiotic preparations on the content of serumcytokines in mice infected with H7N9 avian influenza virus (pg/ml)Bifidobacterium Bifidobacterium B. animalis + Group PBS controlpseudolongum animalis B. pseudolongum Post TNF-α 1.403 ± 0.261 1.349 ±0.403 1.291 ± 0.306 1.434 ± 0.347 infection IL-1β 46.100 ± 0.735  45.834± 0.472  45.889 ± 0.558  46.116 ± 0.297  0 day IL-6 6.373 ± 0.822 6.037± 0.673 5.657 ± 0.946 5.931 ± 0.617 IL-10 3.644 ± 0.559 3.647 ± 0.5563.541 ± 0.538 3.509 ± 0.381 IFN-γ 1.619 ± 0.622 1.651 ± 0.613 1.901 ±0.649 1.659 ± 0.591 IFN-β 31.734 ± 3.116  30.539 ± 2.317  31.040 ±2.853  31.723 ± 3.401  3 days TNF-α 1.234 ± 0.324 1.314 ± 0.413 3.366 ±0.973 4.807 ± 0.990 post IL-1β 53.246 ± 2.708  47.781 ± 2.650  50.884 ±2.052  45.870 ± 2.393  infection IL-6 21.691 ± 5.281  20.364 ± 5.344 25.011 ± 5.501  69.451 ± 12.797 IL-10 3.377 ± 0.193 3.384 ± 0.377 3.899± 0.882 5.243 ± 0.733 IFN-γ 1.750 ± 0.577 1.404 ± 0.300 21.186 ± 13.07958.720 ± 29.046 IFN-β 31.292 ± 3.087  30.032 ± 2.748  37.675 ± 3.562 40.666 ± 3.412  5 days TNF-α 1.321 ± 0.212 1.413 ± 0.282 2.006 ± 0.4831.571 ± 0.426 post IL-1β 65.026 ± 3.797  65.359 ± 3.093  60.184 ± 3.798 58.967 ± 2.768  infection IL-6 54.333 ± 14.122 45.080 ± 16.129 35.990 ±11.465 10.583 ± 4.714  IL-10 3.583 ± 0.714 3.369 ± 0.549 3.896 ± 0.8363.240 ± 0.716 IFN-γ 3.306 ± 0.553 3.099 ± 0.844 8.273 ± 1.693 7.144 ±1.122 IFN-β 35.080 ± 6.752  41.950 ± 7.062  42.102 ± 5.712  52.002 ±6.496 

What is claimed is:
 1. A method for treating or preventing avianinfluenza virus H7N9 infection, comprising preparing a medicinecomprising Bifidobacterium animalis ATCC Accession No. 25527, andapplying the medicine on a subject in need of treatment or preventionfor avian influenza virus H7N9 infection, wherein an effective bacterialconcentration of Bifidobacterium animalis in the medicine is ≥1×109CFU/ml.
 2. The method according to claim 1, wherein the medicine is usedfor improving weight loss caused by avian influenza virus H7N9infection, reducing deaths caused by avian influenza virus H7N9infection, improving pathological injury to lungs caused by influenzavirus infection, or inhibiting proliferation of avian influenza virusH7N9 in lungs.
 3. A medicine for treating or preventing avian influenzavirus H7N9 infection, comprising Bifidobacterium animalis ATCC AccessionNo. 25527, and Bifidobacterium pseudolongum ATCC Accession No. 25526,wherein a ratio of effective bacterial concentration of theBifidobacterium animalis ATCC Accession No. 25527 to the Bifidobacteriumpseudolongum ATCC Accession No. 25526 is 1:0.5 to 1:2, and an effectivetotal bacterial concentration in the mixture of Bifidobacterium animalisATCC Accession No. 25527 and Bifidobacterium pseudolongum ATCC AccessionNo. 25526 is ≥1×109 CFU/ml.
 4. A method for using the medicine asdescribed in claim 3, comprising applying the medicine as described inclaim 3 to a subject in need of treatment or prevention for avianinfluenza virus H7N9 infection.
 5. The method according to claim 4,wherein the medicine is used in the subject for improving weight losscaused by avian influenza virus H7N9 infection, reducing deaths causedby avian influenza virus H7N9 infection, improving pathological injuryto lungs caused by influenza virus infection, or inhibitingproliferation of avian influenza virus H7N9 in lungs.
 6. A method forusing the medicine as described in claim 3, comprising applying themedicine in preparation of an interferon stimulant for treating asubject after infection with avian influenza virus H7N9.